A Novel Dual Allosteric Activation Mechanism of Escherichia coli ADP-Glucose Pyrophosphorylase: The Role of Pyruvate

Abstract

Fructose-1,6-bisphosphate activates ADP-glucose pyrophosphorylase and the synthesis of glycogen in Escherichia coli. Here, we show that although pyruvate is a weak activator by itself, it synergically enhances the fructose-1,6-bisphosphate activation. They increase the enzyme affinity for each other, and the combination increases V max, substrate apparent affinity, and decreases AMP inhibition. Our results indicate that there are two distinct interacting allosteric sites for activation. Hence, pyruvate modulates E. coli glycogen metabolism by orchestrating a functional network of allosteric regulators. We postulate that this novel dual activator mechanism increases the evolvability of ADP-glucose pyrophosphorylase and its related metabolic control.

Structural analysis reveals a pyruvate-binding activator site in the Agrobacterium tumefaciens ADP–glucose pyrophosphorylase

“…We recently found that pyruvate plays a relevant role in the regulation of the enzyme from E. coli (11,23). This was previously overlooked because its synergistic effect was only detected in the presence of the main activator, FBP (11,23). Possibly other enzymes in this family follow a similar trend, and the structural information provided in this work will serve for better predictions.…”

“…It is not clear how many enzymes from the ADP-Glc PPase family are activated by pyruvate, which is critical evolutionary information and therefore deserving of further investigation. We recently found that pyruvate plays a relevant role in the regulation of the enzyme from E. coli (11,23). This was previously overlooked because its synergistic effect was only detected in the presence of the main activator, FBP (11,23).…”

“…However, it is not clear how many of those activators bind to the same promiscuous site or to different sites. At least in E. coli, kinetic data suggested that there must be two synergistic activator sites for fructose 1,6-bisphosphate (FBP) and pyruvate (11). Pioneer protein chemistry and site-directed mutagenesis experiments suggested that the regulatory site was near the C terminus in plant and cyanobacterial enzymes (12)(13)(14) but near the N terminus in nonphotosynthetic bacteria (15,16).…”

“…Pioneer protein chemistry and site-directed mutagenesis experiments suggested that the regulatory site was near the C terminus in plant and cyanobacterial enzymes (12)(13)(14) but near the N terminus in nonphotosynthetic bacteria (15,16). Despite the fact that the exact location of the regulatory site(s) remained elusive for a long time, more recent studies have suggested that the activator-binding site in this family likely resides between the distinct C-and N-terminal domains (5)(6)(7) and that the C-terminal domain likely has more influence on the specificity for pyruvate (11,17). It remains critical to know exactly where the activators bind and how the allosteric signal is transmitted to further understand how glycogen synthesis in bacteria and starch synthesis in plants are controlled.…”

Regulatory Properties of the ADP-Glucose Pyrophosphorylase from the Clostridial Firmicutes Member Ruminococcus albus

“…Previous works showed the interaction between activators and inhibitors of the prokaryotic ADP-Glc PPase (8,11,16,20,21). Thus, we analyzed the activity of the R. albus GlgC/GlgD enzyme in the presence of saturating and nonsaturating concentrations of its effectors (shown in Fig.…”